1. Field of the Invention
The present invention relates to the determination of the relative amount, and copy number, of a particular nucleic acid segment in a biological sample. The invention is particularly useful for determining the viral load, or the number of copies of a viral genome per cell, in a biological sample. The method is therefore especially applicable in the field of medical diagnostics but can also be applied in the fields of genetics, molecular biology, biochemistry, and forensics.
2. Description of Related Disclosures
U.S. Pat. Nos. 4,683,195 and 4,683,202 disclose methods for carrying out PCR, a nucleic acid amplification method, and for using PCR in the detection of specific nucleotide sequences PCR has been automated; the apparatus for automated PCR is disclosed in Ser. No. 899,061, filed Aug. 22, 1986, now abandoned, which is a continuation-in-part of Ser. No. 833,368, filed Feb. 25, 1986, now U.S. Pat. No. 5,331,586. The DNA polymerase of Thermus aquaticus (called Taq polymerase) is especially preferred for PCR; methods for purification and recombinant expression of Taq polymerase are disclosed in Ser. No. 143,441 filed Jan. 12, 1988, now abandoned, which is a continuation-in-part of copending Ser. No. 063,509, filed Jun. 17, 1987, which issued as U.S. Pat. No. 4,889,818, which is a continuation-in-part of copending Ser. No. 899,241, filed Aug. 22, 1986, now abandoned. Methods for structure-independent PCR using 7-deazaguanine are disclosed in copending Ser. No. 248,556, filed Sep. 23, 1988. Methods for inverse PCR are disclosed in copending Ser. No. 203,000, filed Jun. 6, 1988, now abandoned. PCR can be used to detect viral sequences in a sample, as disclosed in Ser. No. 934,955, filed Nov. 26, 1986, now abandoned, which is a continuation-in-part of copending Ser. No. 935,581, filed Nov. 26, 1986, now abandoned in favor of continuation application Ser. No. 394,276, which issued as U.S. Pat. No. 5,008,182. Ser. No. 935,581 is a continuation-in-part of copending Ser. No. 818,127, filed Jan. 10, 1986, now abandoned. Other viral detection methods utilizing PCR are disclosed in Ser. No. 935,271, filed Nov. 26, 1986, now abandoned, and copending Ser. No. 234,486, filed Sep. 9, 1988 now abandoned. DNA preparation solutions preferred for PCR are described in Ser. No. 178,202, filed Apr. 6, 1988, now abandoned. The disclosures of these related patents and publications are all incorporated herein by reference. European Patent Office Publication (EPO) No. 258,017 describes Taq polymerase, a preferred DNA polymerase for use in PCR. PCR has been used to detect the presence of vital nucleic acid sequences, as is disclosed in EPO Nos. 229,701 and 269,445. PCR is also especially useful for determining HLA type, as disclosed in EPO 237,362. Other HLA typing methods are disclosed in U.S. Pat. Nos. 4,582,788 and 4,683,194.
There is still a need in the art, however, for accurate and reliable methods for determining the amount and copy number of nucleic acid sequences in a sample, especially in relation to the copy number of another nucleic acid sequence in the sample. This determination can be of vital importance. For instance, the viral load of an individual, essentially a ratio of vital to individual genomic nucleic acid, can reveal whether a patient is responding to therapy. In the case of the viruses responsible for a disease such as AIDS, the importance of this determination cannot be overstated. The present invention provides a solution to this vital need.